| Spinal cord injury (SCI) is structural and/or functional change in spinal cord due to trauma, fall, and athletic injury, etc. The induced secondary cascade injury, waterfall effect, will lead to secondary damage, which is an important factor causing disability or even death. Therefore, blocking secondary damage in early stage is critical for prognosis of acute spinal cord injury.Many methods are used for spinal cord injury treatments. However, due to difference in spinal cord injuries and complexity of clinic spinal cord injury, most of them are still at the stage of laboratory research. How to get complete functional recovery after spinal cord injury is still an serious problem to be solved. The recent clinical practices indicate that acupuncture therapy plays an important role in spinal cord injury treatment. An early acupuncture therapy will stop or alleviate secondary lesion induced by SCI, and promote repair of neurons and neural fibers. This will further accelerate the recovery of SCI, effectively avoid complications, reduce secondary spinal cord damage, and facilitate recovery and regeneration of neural functions. However, its concrete mechanism is unclear. It will be of important scientific significance and pratical application to study the molecular mechanism of spinal cord injury and evaluate the effect of acupuncture therapy at proteome level .By using normal rats as control, the protein two dimensional electrophoresis (2-DE) technique was applied to analyze the expression difference in spinal cord proteome from rats with acute spinal cord injury(ASCI). Thirty rats were divided into two groups, the normal control group and the ASCI model group. The spinal cords from 3 rats were combined into a sample. The experiments were repeated for five times for each sample, The ASCI rats were obtained by the improved Allen's Beat. The rats were hitted on T10～T11 when the hitting energy was 50g/2cm (10g×5cm), so as to cause moderate acute spinal cord injury. The rats were then sacrificed and the spinal cords were separated at certain time. The total spinal cord proteins were extracted and 2-DE was performed. The 2-DE gel images were analyzed by using Image Master 2D Platinum 5.0 software, so as to identify the differential protein expression. Analysis of peptide mass fingerprinting (PMF) and amino acid sequence was conducted on significant protein differential spots by matrix-assisted laser time of flight mass spectrometry desorption/ionization (MALDI-TOF/TOF-MS) and high performance liquid chromatography–electrospray tandem MS NanoUPLC-ESI- MS/MS. The proteins were searched and identified using the Swiss-prot database and MASCOT.1170±62 and 1324±47 protein spots were detected by Imagemaster analysis software in the 2-DE gel images from normal rats and ASCI rats, respectively. The match rate is about 79.5% for spectrum profiles of normal rats and ASCI rats. Totally 31 significant protein spots were identified, including 9 up-regulated spots, 20 down-regulated spots, and 2 new spots. The pI of the differential spots are in 4.0-9.2, molecular weights rang from14.0Kda to 100.0Kda, the up-regulated expressions are in 3-25 times, and the lowest down-regulated expression is 0.1 times.In the same way, forty-five rats were divided into three groups, the 6 hours, 24 hours and 48 hours electroacupuncture therapy group. The spinal cords from 3 rats were combined into a sample, the experiments were repeated for five times in each sample, using the spinal cord from normal rats as controls, differential proteins in ASCI rats and electroacupuncture treated ASCI rats at 6 hours, 24 hours, and 48 hours after treatment were identified. As compare to ASCI rats, 1345±57, 1293±39 and 1216±76 protein spots were detected by Imagemaster analysis software at 6 hours, 24 hours, and 48 hours after electroacupuncture therapy, respectively. The match rates are among 74.9%~85% for spectrum profiles of ASCI rats. Differential proteins could be classified as 4 groups: 1. "slightly decreasing type ", protein spot 1, 2, 5 and 6, should be the candidate hints for cure or effective. 2. " sharply decreasing type ", protein spot 7, 8 and 9, might be the indicators for the effectiveness of electroacupuncture therapy. 3. " fluctuation type " protein spot 10 , 23 and 37, might indicated the complicated mechanism in damage repair. 4. " sharply increasing type ", protein spots 4 and 17, might be the candidate hints for cure or effective.10 protein spots with higher abundance were analyzed by MALDI- TOF/TOF-MS, and 13 significant proteins were identified. The Mascot search score was 103-506, peptide matching number was 2~9 and sequence coverage was 14~59%. 21 protein spots with lowwer abundance were analyzed by MS NanoUPLC- ESI-MS/MS technique, and 29 significant proteins were identified. The Mascot search score was 57~805, peptide matching number was 2~22 and sequence coverage was 4~70%.13 proteins by identified in the 9 up-regulated protein spots include NADH dehydrogenase, cytosolic malate dehydrogenase(cMDH), putative nucleoside diphosphate kinase(NDK), Nm23 protein , Myelin basic protein(MBP),ubiquitin- conjugating enzyme E2N(UBE2N), DJ-1 protein, similar to CAP1 protein, triosephosphate isomerase 1 ( TPI1 ) , phosphoglycerate mutase ( PGM ) , glyceraldehydes-3-phosphate dehydrogenase(GAPDH), ubiquitin carboxy-terminal hydrolase L1(UCH-L1), Rho GDP dissociation inhibitor(GDI)and the spot 3 are the mixture of NDK and Nm23, the spot 4 are the mixture of MBP and UBE2N, the spot 5 are the mixture of DJ-1 and CAP1. The spot 13 are the mixture of UCH-L1 and GDI.27 proteins by identified in the 20 down-regulated protein spots include proteasome, ATP synthase, isocitrate dehydrogenase 3, guanine nucleotide-binding protein beta 2 subunit, G protein beta 1 subunit, Cu-Zn superoxide dismutase, phosphatidylethanolamine binding protein, peroxiredoxin 2, lactate dehydrogenase,sirtuin, aldehyde dehydrogenase 2, unnamed protein product, dihydrolipoamide S-succinyltransferase, Hnrph1 protein , N-myc downstream regulated gene 1, adenylate kinase isozyme 1, carbonic anhydrase 1,3-oxoacid CoA transferase 1, cyclase-associated protein, glial fibrillary acidic protein, es1 protein, lpha B-crystallin, 3-phosphoglycerate dehydrogenase,aldehyde reductase 1, M2 pyruvate kinase, quinoid dihydropteridine reductase and triosephosphate isomerase.The both of the 2 new protein spots are Neurofilament medium polypeptide (NF-M).12 differential expression protein spots were identified to be related to the EA therapy for ASCI, 15 proteins identified including NADH dehydrogenase, Cytosolic malate dehydrogenase, Myelin basic protein, ubiquitin-conjugating enzyme E2N, DJ-1 protein, cyclase-associated protein, triosephosphate isomerase 1, phosphoglycerate mutase, Neurofilament medium polypeptide, glyceraldehyde -3-phosphate dehydrogenase, Cu-Zn superoxide dismutase, Sirtuin, 3-oxoacid CoA transferase 1, cyclase- associated protein1 and glial fibrillary acidic protein(GFAP). It was reported that MBP, NF-M, GFAP and SOD1 were closely related to spinal cord injury. and the expression level of NADH dehydrogenase, DJ-1 protein, triosephosphate isomerase, proteasome,peroxiredoxin 2, N-myc downstream regulated gene 1,guanine nucleotide-binding protein beta 2 subunit, phosphoglycerate mutase, Sirtuin, ATP synthase,adenylate kinase isozyme 1 and phosphatidylethanolamine binding protein will be changed in spinal cord injury.There is not any report about the role of other 25 proteins in ASCI. The newly discovered differential proteins will provide valuable clues for further studies onpathogenic and repair mechanism of ASCI and functional mechanism of EA on ASCI.
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